The long term objectives of the proposed research are to understand the basic mechanisms of pre-messenger RNA (pre-mRNA) splicing, and the mechanisms by which alternative splicing is regulated. A primary focus of the proposed studies will be on the role of specific protein-protein and protein-RNA interactions in both processes. With regards to basic splicing mechanisms, experiments will be carried out to investigate the nature of specific interactions between individual members of the serine/arginine rich (SR) family of splicing factors and other components of the mammalian spliceosome. This analysis will involve the use of coimmunoprecipitation methods, and the use of a yeast in vivo protein-protein interaction assay. The interaction domains of SR proteins and their specific partners will be identified and characterized. Once identified, the domains in different proteins will be exchanged, and the functional consequences determined by in vitro splicing. Overall, this line of investigation will be directed towards testing the hypothesis that SR proteins play an essential role in splice site selection by mediating interactions between splicing factors bound to the 5' and 3' splice sites. The relationship between interactive components of the spliceosome will also be investigated using protein crosslinking methods with purified prespliceosomes and spliceosomes. These experiments are designed to address the possibility that dynamic patterns of protein protein interactions occur during spliceosome assembly and these interactions may be controlled by protein phosphorylation/dephosphorylation. The regulation of alternative splicing will be investigated by taking a multidirectional approach to the analysis of a splicing enhancer complex that is assembled on a regulatory sequence present in the Drosophila doublesex pre-mRNA. The role of SR proteins and the regulatory proteins transformer and transformer2 (Tra and Tra2) in recruiting spliceosome components in to this complex will be investigated. In addition, novel components of the complex will be identified, purified using an affinity purification method, and ultimately cloned. A variety of methods will be used to study the structure of the RNA within the complex and to identify specific protein-protein and protein-RNA interactions. Finally, effort will be made to identify novel splicing enhancers by virtue of their ability to specifically interact with SR proteins in the presence of nuclear extract. The objective of these studies is to determine whether splicing enhancers are used widely in both splice selection and regulated alternative splicing. If successful, these studies should provide significant advances in our understanding of the mechanisms of pre-mRNA splicing. The accurate removal of introns from pre-mRNA is required for normal development and cell division. Because many forms of human genetic disease and certain forms of cancer are a consequence of aberrant splicing, an understanding of the process is directly relevant to human health .